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Scientists working with recombinant proteins expressed in Escherichia coil probably use at least one liquid chromatography technique to purify their protein of interest. But liquid chromatography frequently requires a considerable amount of optimization, and usually involves several different chromatographic steps to rid the sample of contaminants. (1) The ideal solution would be to create a resin that is completely specific to the target protein, enabling one-step purification.
Affinity chromatography theoretically does just that--a ligand that specifically interacts with the target protein is immobilized on a chromatography matrix; the target protein binds to the column, and unwanted proteins are eluted. In some cases, the affinity ligand is an antibody against the protein of interest; in others, the target protein is expressed from a plasmid that encodes for an "affinity tag" specific to a particular ligand.
This article discusses some of the affinity fusion systems available for recombinant proteins expressed in E. coil A list of companies offering complete affinity fusion protein-purification systems (vectors and resins) is shown in the accompanying table.
IMAC-CULATE PURIFICATION One of the most common, and oldest, affinity purification methods uses glutathione S-transferase, or GST, tags. These fused proteins are easily purified using a glutathione-coupled matrix, but the system has several disadvantages. First, purification requires that the GST domain be properly folded. Further, the tag's large size--220 amino acids--can both hinder the solubility of the expressed protein, leading to. formation of inclusion bodies, and distort the protein's native conformation, making structural studies difficult. As a result, the GST tag is often removed after purification, usually via a cleavage site engineered between the tag and the target protein.
Although the GST tag is more difficult to use than its popular competitor, the polyhistidine tag, many protein chemists prefer it because its use has been so well documented. "The six-His tag has become very popular, and a lot of scientists choose it, but ... a lot of people are still using GST because they are used to it," explains Krishna Mallia, a scientist at Rockford, Ill.-based Pierce Chemical Co. (www.piercenet.com). Companies offering GST purification systems and products include Pierce, Piscataway, N.J.-based Amersham Biosciences, and Madison, Wis.-based Novagen.
In recent years usage of polyhistidine fusion tags has eclipsed that of GST tags. First described by Jerker Porath and colleagues in the mid-1970s, this technique, called IMAC (immobilized metal ion affinity chromatography), exploits the affinity of histidine's imidazole side chains for metal ions such as nickel, zinc, and cobalt. (2,3) IMAC generally uses tags composed of six histidine residues (6xHis) and a nickel-based affinity resin. His-tagged proteins adsorb to the column under neutral to alkaline pH conditions and are eluted at low pH or by competitive adsorption with imidazole.
Pierce's Mallia explains that the 6xHis tag has grown in popularity over the GST fusion tag, primarily because of its small size, which means the tag does not usually need to be cleaved from the fusion protein. Also, unlike GST-based purification systems, researchers can purify 6xHis fusions under denaturing conditions. Finally, the 6xHis tag is relatively non-immunogenic, a benefit when scientists inject the purified proteins into live animals.
Several companies sell complete IMAC systems. Valencia, Calif.-based QIAGEN's QIAexpress system uses an [Ni.sup.2+]-nitrilotriacetic acid (Ni-NTA) resin and offers vectors to fuse the tag to either the recombinant protein's N- or C-terminus. Novagen's His Bind[R] Purification Kits are similar, allowing purification of fusion proteins with six-to-10-histidine-residue-long tags. Novagen offers Ni-NTA- and IDA (iminodiacetic acid)-based resins on a variety of supports, including bulk agarose, magnetic agarose, and Fractogel[R].
Carlsbad, Calif.-based Invitrogen's Xpress[TM] System is also a 6xHis-tag purification system. Its expression vectors, pTrcHis, encode for an enterokinase cleavage site to remove N-terminal tags. The company's ProBond[TM] metal affinity resin, included in the system, can be used for both gravity flow chromatography and FPLC. In contrast, QIAGEN's Ni-NTA resin comes in two different forms--one for gravity flow called Ni-NTA Agarose, and one with a more highly crosslinked support called Ni-NTA Superflow for FPLC.
Despite its many advantages, polyhistidine tags also present several pitfalls, says Yamuna Dasarathy, marketing manager at Amersham Biosciences, which offers affinity resins for purification of His-tagged proteins as well as fusion vectors and affinity resins for purification of GST-tagged proteins. These problems include the formation of inclusion bodies, difficulty in solubilization, lack of stability, and incorrect refolding of the fusion proteins. Further, nickel-based resins may be prone to leakage of metal ions, which can remain in the protein solution and damage the amino acid side chain of the target …